Electron Microscopy Reveals Graded Ultrastructural Disruption from Microsecond Electroporation Pulses

In a comparative ultrastructural investigation evaluating microsecond pulsed electric fields, researchers have demonstrated that electroporation triggers a graded morphological response in cellular membranes and subcellular organelles. Across both cultured tumor cells and animal tissue models, electrical stimulation manifested as distinctly different structural outcomes depending directly on pulse intensity, producing relatively limited, survivable intracellular changes under operational reversible conditions while generating severe, widespread membrane disruption and organelle destruction under operational irreversible protocols.

The study addressed a persistent gap in biomedical engineering and cellular biophysics, where direct microscopic comparisons between reversible electroporation (RE) and irreversible electroporation (IRE) have remained sparse despite widespread interest in their respective biological consequences. Although electric pulse parameters and cellular environments are known to determine whether plasma membrane permeabilization resolves reversibly or transitions toward irreversible breakdown, the precise multilevel structural phenotypes associated with these distinct electrical thresholds had not been comprehensively contrasted under uniform experimental conditions. To establish this morphological framework, the investigators evaluated structural alterations across multiple scales in cancer cells and mammalian organ tissues.

The experimental design focused on two distinct cultured malignant cell lines: KPC pancreatic cancer cells and Hepa1-6 hepatocellular carcinoma cells. To administer the microsecond electric fields, the authors applied a standardized sequence of sixty electrical pulses, with each pulse delivering a duration of 90 microseconds. The operational protocol for reversible electroporation delivered an electric field strength of 750 volts per centimeter across the cell suspensions. In contrast, the operational protocol for irreversible electroporation maintained the identical pulse count and pulse duration while elevating the electric field intensity to 1,250 volts per centimeter, establishing the operational benchmark for high-field irreversible intervention.

To assess the structural and functional consequences of the two pulsed field regimes, the research team deployed multiple complementary analytical modalities. Transmission electron microscopy (TEM) served as the primary imaging method for resolving the internal ultrastructure and morphology of mitochondria and the endoplasmic reticulum. Scanning electron microscopy (SEM) was utilized to examine the topography and outer surface architecture of the plasma membrane. Membrane permeabilization was evaluated functionally through cellular uptake assays employing free fluorescein isothiocyanate (FITC). To characterize discrete nanoscale membrane defects, candidate nanopores were operationally defined using control-derived diameter thresholds and subsequently evaluated through Gaussian mixture modeling.

Under the 750 V/cm operational reversible electroporation protocol, transmission electron microscopy identified a defined profile of intracellular organelle alterations. Mitochondria in treated tumor cells exhibited morphological contraction accompanied by an increase in mitochondrial matrix electron density and a measurable reduction in internal cristae. At the level of the endoplasmic reticulum, the cellular response to reversible electrical pulsing was characterized by limited, contained luminal dilation without overt structural fragmentation. These intracellular organelle changes occurred under field conditions where the plasma membrane retained its operational capacity for preservation rather than catastrophic disintegration.

In sharp contrast to the moderate alterations observed following reversible treatment, operational irreversible electroporation at 1,250 V/cm produced severe, extensive damage across cellular structures. Cells exposed to these high-field pulses exhibited pronounced mitochondrial swelling, extensive dilation and swelling of the endoplasmic reticulum, widespread disruption of mitochondrial cristae, and catastrophic fragmentation of cellular membranes. Functionally, this profound structural damage correlated with a marked elevation in free-FITC uptake, verifying lost membrane barrier integrity. On the external cell boundary, scanning electron microscopy revealed the emergence of numerous distinct membrane surface depressions across both tested tumor models.

Quantitative morphological measurements revealed substantial differences in surface depression sizing between the two cell lines after irreversible electroporation. Statistical evaluation demonstrated that irreversible pulses shifted the median membrane depression diameter from 28 nanometers to 48 nanometers in KPC pancreatic cancer cells, while in Hepa1-6 hepatocellular carcinoma cells, the median diameter expanded from 30 nanometers to 111 nanometers, with both shifts achieving statistical significance (Holm-adjusted P < 0.001). Furthermore, Gaussian mixture modeling of candidate-nanopore diameters revealed non-unimodal, cell line-specific distributions. In KPC cells, the modeled candidate nanopores segregated into fitted components centered at approximately 91 and 262 nanometers, whereas in Hepa1-6 cells, the fitted distribution resolved into three distinct components centered at approximately 101, 199, and 369 nanometers.

To verify whether the structural phenomena observed in vitro corresponded to tissue-level outcomes in a large-animal model, the investigators analyzed porcine liver and pancreas specimens collected 24 hours after the delivery of irreversible electroporation pulses. In these post-treatment animal tissues, electron microscopy identified tissue surface alterations closely related to the membrane perturbations seen in culture. Furthermore, the examination of vascular spaces within the targeted porcine organs revealed discrete, pore-like membrane discontinuities on erythrocytes, documenting that microsecond irreversible electric fields generate membrane defects in circulating red blood cells within treated in vivo tissue beds.

The investigators concluded that microsecond electroporation produces a continuous, graded ultrastructural spectrum that progresses from limited, contained organelle restructuring during reversible stimulation to widespread membrane fragmentation and organelle disruption during irreversible pulsing. By elucidating these specific nanoscale surface depressions, organelle swelling patterns, and species- and tissue-level morphological responses, the study establishes a foundational structural framework. This evidence-based ultrastructural baseline provides key benchmark parameters to support future mechanistic investigations and guide the systematic optimization of electrical pulse protocols in biomedical engineering applications.

Subject of Research: Ultrastructural cellular and tissue alterations induced by reversible and irreversible electroporation

Article Title: Electron microscopy characterization of ultrastructural alterations induced by reversible and irreversible electroporation

Article References: Ma, W., Xu, L., Zhang, C., Xu, F., Lou, W., Liu, H., Xie, L., & Jiang, T. (2026). Electron microscopy characterization of ultrastructural alterations induced by reversible and irreversible electroporation. BioMedical Engineering OnLine. https://doi.org/10.1186/s12938-026-01649-9

Image Credits: AI Generated

DOI: 10.1186/s12938-026-01649-9

Keywords: electroporation, reversible electroporation, irreversible electroporation, transmission electron microscopy, scanning electron microscopy, ultrastructure, candidate nanopores, mitochondrial morphology, endoplasmic reticulum, membrane permeability, KPC cells, Hepa1-6 cells, porcine liver, porcine pancreas, FITC uptake, Gaussian mixture modeling